Thymic differentiation involves a discrete series of stages leading to positive and negative selection. Much is known regarding changes in the cell surface phenotype during T cell maturation. By contrast, little is understood regarding the intracellular signaling mechanisms that control differentiation. The applicant has previously reported the identification of a 120/130 kDa protein that binds to the SH2 domain of the src-family tyrosine kinase Fyn. The applicant believes that these polypeptides correspond to CAS, an adapter protein with multiple YDXP motifs and a single SH3 domain. These pYDXP motifs are ideally suited to bind to the SH2 domain of the intracellular protein CRK. Recently, the applicant has found that thymocytes exhibit a highly restricted pattern of tyrosine phosphorylation with only two major sets of phosphorylated substrates at 120/130kDa and 50-65kDa, in contrast to mature T cells, where the pattern is much more complex. Anti-120/130 antibodies immunoprecipitated the majority of the prominently tyrosine phosphorylated polypeptides from thymocytes. This suggests that CAS and associated proteins make up the majority of tyrosine phosphorylated proteins in thymocytes. This observation suggests that CAS and associated proteins may play important roles in thymic differentiation. In this application, the investigator proposes to study further the role of CAS and associated proteins in signaling in thymocytes and mature T cells by exploring the interaction between CAS, CRK, and the guanine nucleotide releasing factor C3G. C3G may be involved in the conversion of Ras to a GTP-bound active state, an event that is central to the control of cellular growth and differentiation.
In Aim 2, the applicant will attempt to isolate a cDNA encoding a T-cell specific form of CAS. An antisera generated against p120/130 isolated from T cells is restricted in its recognition of the T cell form, termed CAS-T.
In Aim 3, the applicant will transfect a T cell hybridoma DC.27 with forms of CAS or CAS-T for analysis of its role in production of IL-2. Lastly, in Aim 4, the applicant will generate CAS or CAS-T knockout mice for analysis of the role of CAS in thymic development.
